Membrane transport proteins are a large class of integral membrane proteins involved in the movement of small molecules across cellular membranes. Many of these proteins, known as secondary active transporters, use pre-existing substrate concentration gradients as an energy source for translocating another substrate against its concentration gradient. Every organism expresses dozens of different secondary transporter proteins with a diverse array of structural folds, and each protein is specific for a different substrate, which range from ions to neurotransmitters. A detailed understanding of the mechanism of each membrane transport protein requires knowledge of its three-dimensional structure in a number of different conformational states and identification of the binding regions for the substrate or substrates. We have identified a novel fold for a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, based on an analysis of repeated elements in its protein sequence, and by comparison with structures of known proteins (1). We identified a template for NaPi-IIa within a substructure of a distantly-related protein, and used state-of-the-art sequence alignment strategies and homology modeling to construct a three-dimensional model of the fold of NaPi-IIa. The model was compared with available experimental data on its transmembrane topology, finding excellent agreement. We then predicted binding sites for two of the three required sodium ions and for the phosphate group; these predictions were tested by site-specific mutagenesis of judicious positions in the predicted binding site of NaPi-IIa that was mutated at judicious positions in the predicted sites, by electrophysiology and uptake measurements (carried out by the Forster lab, Zurich), providing strong support for the predictions (1). Secondary transporters cycle through a number of different conformational states during transport, and therefore it is critical to understand to which particular states the substrates or co-substrates are bound. Our collaborators (Ziegler lab, MPI Frankfurt/University Regensburg) solved structures of a sodium-coupled betaine transporter (BetP) whose structural fold is similar to the neurotransmitter:sodium transporters (NSS) - where the protein clearly adopts different states. In two distinct outward-facing conformations, the presence and absence of the substrate (choline, in this case) could be clearly distinguished, but the presence of sodium was unclear (2). We used molecular dynamics simulations to characterize the sodium occupancy of these states, finding that both choline-bound and choline-free outward-facing conformations are likely to be occupied by sodium (2). Moreover, the binding sites exhibited differing degrees of solvation, indicating a progressive ion dehydration mechanism upon choline binding, and upon the conformational change toward the occluded and closed states (2). The levels of neurotransmitter in neuronal synapses is regulated by the amount of neurotransmitter transporter that is present in the neuronal membrane, which in turn is modulated by interactions with cytoplasmic proteins such as syntaxin 1A. Serotonin uptake by serotonin transporter (SERT), for example, is regulated through interactions involving its N- and C-terminal domains, whose structures have been difficult to characterize using crystallography. We carried out a large-scale modeling study (3) to predict the folds of the (83-residue) N-terminal domain and the (60-residue) C-terminal domain of human SERT using the fragment-based structure prediction technique, Rosetta. Specifically, we constructed one million candidate folds per terminal domain, which were then clustered by structural similarity, and filtered based on results from FRET measurements from our collaborators (Sitte lab, University of Vienna). The secondary structure composition of the SERT terminal domain models agreed well with results from proton NMR measurements (Konrat lab, University of Vienna), as well as circular dichroism spectroscopy on constructs of the N-terminal domain (Konrat lab, University of Vienna; Singh lab, Yale University) (3). We conclude that the SERT terminal domains contain structured elements, albeit sometimes separated by unstructured (intrinsically disordered) segments. We combined candidate folds with template-based models of the transmembrane domains to construct full-length models of SERT and analyzed the probable locations and separation of the two domains in two different conformational states. Using these low-resolution, full-length models, hypotheses for the interactions of SERT with regulatory proteins were derived (3).